Stability and biological activity of Merlot (Vitis vinifera) grape pomace phytochemicals after simulated in vitro gastrointestinal digestion and colonic fermentation

Journal of Functional Foods 36 (2017) 410–417

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Stability and biological activity of Merlot (Vitis vinifera) grape pomace phytochemicals after simulated in vitro gastrointestinal digestion and colonic fermentation Rúbia C.G. Corrêa a,b,c, Charles W.I. Haminiuk a,⇑, Lillian Barros c, Maria Inês Dias c,d, Ricardo C. Calhelha c, Camila G. Kato b,e, Vanesa G. Correa b,e, Rosane M. Peralta b,e, Isabel C.F.R. Ferreira c,⇑ a

Graduate Program in Food Technology, Federal University of Technology - Paraná (UTFPR), Campus Campo Mourão, Paraná, Brazil Department of Biochemistry, State University of Maringá, Paraná, Brazil c Mountain Research Centre (CIMO), ESA, Polytechnic Institute of Bragança (IPB), Campus de Santa Apolonia, Bragança, Portugal d Laboratory of Separation and Reaction Engineering - Laboratory of Catalysis and Materials (LSRE-LCM), Polytechnic Institute of Bragança, Campus de Santa Apolónia, Bragança, Portugal e Graduate Program in Food Science, State University of Maringá, Paraná, Brazil b

a r t i c l e

i n f o

Article history: Received 7 April 2017 Received in revised form 10 July 2017 Accepted 11 July 2017

Keywords: Merlot grape pomace In vitro gastrointestinal digestion In vitro fermentation Phenolic compounds Antiproliferative effects

a b s t r a c t Grape pomace is an abundant/accessible food industry by-product that contains a wide range of phenolic compounds, which have been related to several health benefits and bioactivities. The aim of this study was to mimic the gastrointestinal digestion and the colonic fermentation of Merlot grape pomace, in order to unravel possible phytochemical contents reductions and the processes associated with them, as a tentative to relate the phenolic compound profiles of the extracts with their biological properties. LC-DAD-ESI/MS suggested that the in vitro digestion process promoted drastic qualitative and quantitative reductions in the phenolic compounds profile of the Merlot grape pomace crude extract. Such alterations could be related to the decreases of some bioactivities of the extract, which seems to be the case of antioxidant and antibacterial properties, although not in a directly proportional manner. However, the simulated colonic fermentation seems to have a positive effect over the extract’s antiproliferative potential. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Winemaking is currently one of the most relevant agro-industrial activities in the world. Undoubtedly, grapes are an abundant fruit crop worldwide, being Vitis vinifera the species most frequently cultivated for wine production (Otero-Pareja, Casas, Fernández-Ponce, Mantell, & Ossa, 2015; Barba, Zhu, Koubaa, Sant’Ana, & Orlien, 2016). The co-products generated by the vitiviniculture sector activities, such as pomace, rachis, and lees, corresponds to incredible 30% of the total amount of vinified grapes (Makris, Boskou, & Andrikopoulos, 2007), most of them still underexplored and commonly discarded without adequate treatment, which leads to environmental impact (Melo et al., 2015). Several studies have already proved that these winemaking co-products constitute an interest-

⇑ Corresponding authors. E-mail addresses: [email protected] (C.W.I. Haminiuk), [email protected] (I.C.F.R. Ferreira). http://dx.doi.org/10.1016/j.jff.2017.07.030 1756-4646/Ó 2017 Elsevier Ltd. All rights reserved.

ing source of natural antioxidants, especially phenolic compounds. Grape pomace, the major winery sub-product, consists of the waste seeds, skins and stems that remain after the grape pressing process, characterized by an expressive content of phenolic compounds due to the incomplete extraction that occurs during the winemaking process. (Jara-Palacios et al., 2015; Otero-Pareja et al., 2015). In recent years, there has been an increasing interest in the exploitation of polyphenol-rich winery sub-products to produce novel extracts and health promoting products (Fontana, Antoniolli, & Bottini, 2013). As such, the recovery of phytochemicals from industrial food co-products represents a sustainable and cost effective source of high-value bioactives, which could be recycled and return to the food chain as functional food ingredients and nutraceuticals (Corrêa et al., 2016). Although in the past decade a number of researches have addressed the extraction, chemical characterization and antioxidant capacity of grape pomace extracts from diverse varieties (Makris et al., 2007; Amico, Chillemi, Mangiafico, Spatafora, &

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Tringali, 2008; Rockenbach et al., 2011; Sagdic et al., 2011; Fontana et al., 2013; Doshi, Adsule, Banerjee, & Oulkar, 2015; Iora et al., 2015; Otero-Pareja et al., 2015; Ribeiro et al., 2015), only a few studies have explored their antibacterial (Tseng & Zhao, 2012; Oliveira et al., 2013) and anti-inflammatory properties (Melo et al., 2015). However, there are even fewer researches over the antiproliferative potential of grape pomace extracts (Jara-Palacios et al., 2015). More recently, the influence of the gastrointestinal digestion step on the food phytochemicals contents, and on their bioactivities (mainly antioxidant capacity), have attracted special attention, which is evidenced by the great number of publications dedicated to this theme (Gumienna, Lasik, & Czarnecki, 2011; Tavares et al., 2012; Correa-Betanzo et al., 2014; Pavan, Sancho, & Pastore, 2014; Podswdek et al., 2014; Mosele, Macià, Romero, Motilva, & Rubió, 2015; Wu, Teng, Huang, Xia, & Wei, 2015; Del PinoGarcía, González-SanJosé, Rivero-Pérez, García-Lomillo, & Muñiz, 2016; Kaulmann, Legay, Schneider, Hoffmann, & Bohn, 2016; López-Barrera, Vázquez-Sánchez, Loarca-Piña, & Campos-Vega, 2016). Nonetheless, except for the recent work of Gil-Sánchez et al. (2017), reports on the effects of in vitro digestion and simulated colonic fermentation processes on grape pomace phenolic compounds as well as on its bioactive properties, such as antiproliferative effects and hepatotoxicity, are lacking. In view of the above, the aim of this study was to mimic the gastrointestinal digestion and the colonic fermentation of Merlot grape pomace, in order to unravel possible phytochemical contents reductions and the processes associated with them, as a tentative to relate the phenolic compound profiles of the extracts with the herein assessed bioactivities. For this purpose, the three grape pomace extracts obtained, namely crude, digested and fermented extracts, were characterized in terms of non-anthocyanin and anthocyanin compounds. The antioxidant, antibacterial and antiproliferative potentials of the grape pomace extracts were also evaluated and compared, and the hepatotoxicity was assessed in a primary cell culture of porcine liver cells.

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contamination and fermentation processes, the grape pomace was dried in an air circulation oven at 80 °C during 36 h. The dried mass was therefore milled to a fine powder (40 mesh), transferred to polyethylene film bags under vacuum packing and kept at 20 °C until analysis (Ribeiro et al., 2015). This material constituted the matrix used to obtain three different extracts, according to the process shown in the diagram of Fig. 1. 2.3. Crude extract preparation The grape pomace crude extract was obtained according to the procedure previously described by Ribeiro et al. (2015). The extractions were performed in the ratio 1:50 (m/v—solute/solvent) with ethanol and distilled water (40:60, v/v), respectively. The mixture was stirred for 24 h on a shaker at 25 °C. The tubes containing the solutions were centrifuged at 5000 rpm for 25 min and the supernatant was separated. The obtained filtrate was concentrated with a rotary vacuum evaporator at 40 °C in order to eliminate the solvent, posteriorly freeze-dried and stored at 20 °C until use. 2.4. In vitro digestion The in vitro gastrointestinal digestion was simulated according to methodology described by Koehnlein et al. (2016). Briefly, 13 g of the lyophilized grape pomace hydroethanol extract was mixed

2. Materials and methods 2.1. Standards and reagents Acetonitrile (99.9%) was of HPLC grade from Fisher Scientific (Lisbon, Portugal). Phenolic standards were from Extrasynthèse (Genay, France). Salivary alpha-amylase (6.66 U/mL), pancreatin (100 U/mL), pepsin A (1923 U/mL), bile extract, Sulforhodamine B, trypan blue, trichloroacetic acid (TCA), trolox (6-hydroxy-2,5,7, 8-tetramethylchroman-2-carboxylic acid), formic acid and Tris were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), hank’s balanced salt solution (HBSS), fetal bovine serum (FBS), L-glutamine, trypsinEDTA, penicillin/streptomycin solution (100 U/mL and 100 mg/ mL, respectively) were purchased from Gibco Invitrogen Life Technologies (California, Massachusetts, USA). The tumor cell lines were provided by DSMZ (Braunschweig, Germany). 2,2-Diphenyl1-picrylhydrazyl (DPPH) was obtained from Alfa Aesar (Ward Hill, MA, USA). All other general laboratory reagents were purchased from Panreac Química S.L.U. (Barcelona, Spain). Water was treated in a Milli-Q water purification system (TGI Pure Water Systems, Carrollton, TX, USA). 2.2. Grape pomace The fresh Merlot (Vitis vinifera) grape pomace was donated by a winemaking company located in the State of Paraná, Brazil. Immediately after its obtainment, in order to prevent microbiological

Fig. 1. Diagram of the main steps performed in the obtainment of the Merlot grape pomace extracts and comparative performed assays.

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with 39 mL of artificial saliva solution (2.38 g Na2HPO4, 0.19 g KH2PO4, 8 g NaCl in 1 L of distilled water). The pH was regulated to 6.75, at the temperature of 37 °C and a-amylase was added to produce an enzyme activity of 200 U. This blend was shaken at 150 rpm during 10 min. In sequence, the pH was adjusted to 1.2 and 39 mL of artificial gastric fluid (0.32 g pepsin in 100 mL of 0.03 M NaCl, pH 1.2) was included. The mixture was then incubated on a shaker at 37 °C for 120 min, under agitation of 150 rpm. Lastly, the pH was adjusted back to 6.0 following the addition of 6.5 mL of NaCl (120 mM), 6.5 mL of KCl (5 mM) and 39 mL of artificial intestinal fluid (0.15 g of pancreatin and 0.9 g of bile extract in 100 mL of 0.1 M NaHCO3). The mixture was incubated at 37 °C for 60 min, at 150 rpm. Thereon the obtained digested extract was freeze-dried and stored at 20 °C. 2.5. In vitro colonic fermentation The fermentation medium, prepared according to methodology described by Karppinen, Liukkonen, Aura, Forssell & Poutanen (2000) with modifications, was a carbonate-phosphate buffer. The mineral medium was regulated to pH 7.0 and glucose was added to a final concentration of 0.8%. The mixture was purged with nitrogen until the anaerobic indicator (methylene blue) turned colorless. The inoculum was obtained from fresh feces collected from the entire large intestines of male Wistar rats (75-days old animals, average 250 g) immediately after euthanasia. A fecal pool of 5 animals was made. Immediately after collecting, the material was homogenized with the culture medium at a ratio of 1:10 (w/v). The bottles were bubbled over again with nitrogen and closed airtight. Afterwards, the bottles were incubated at 37 °C for 24 h under agitation of 50 rpm, in order to simulate the condition in the colonic lumen. The initial pH was 7.0 and the final pH was around 5.0. A control with the culture medium and inoculum was prepared. In order to verify the absence of phenolic in the diet, the control sample was submitted to the Folin-Ciocalteu assay, with negative results. Subsequently, the material was ultra-centrifuged at 31,000 rpm during 30 min, sterilized by filtration, and freeze-dried. As phenolic compounds and antioxidant activity were not detected in the control, it was not considered for the antioxidant and bioactive assays. 2.6. Phenolic compounds analysis The phenolic profile was determined by LC-DAD-ESI/MSn (Dionex Ultimate 3000 UPLC, Thermo Scientific, San Jose, CA, USA). The lyophilized extracts were re-dissolved at a concentration of 5 mg/mL with an ethanol:water (40:60, v/v) mixture. The non-anthocyanin compounds were separated and identified as previously described by Bessada, Barreira, Barros, Ferreira, and Oliveira (2016) and the detection was carried out in a DAD (280 and 370 nm as preferred wavelengths) and in a mass spectrometer (MS), operating in negative mode. The anthocyanin compounds were separated and identified as previously described by Gonçalves et al. (2017) and detection was carried out in DAD (520 nm the preferred wavelength) and in a mass spectrometer (MS), operating in positive mode. For both non-anthocyanin and anthocyanin compounds the MS detection was performed using a Linear Ion Trap LTQ XL mass spectrometer (ThermoFinnigan, San Jose, CA, USA) equipped with an ESI source. The identification of the phenolic compounds (non-anthocyanin compounds and anthocyanin compounds) were performed using standard compounds, when available, by comparing their retention times, UV–vis and mass spectra; and also, comparing the obtained information with available data reported in the literature giving a tentative identification. For quantitative analysis, a calibration curve for each available phenolic standard was constructed

based on the UV signal. For the identified phenolic compounds for which a commercial standard was not available, the quantification was performed through the calibration curve of the most similar available standard, such as for compounds 1 and 3–8 were quantified (+)-catechin (y = 84950x 23200; R2 = 0.999), compound 2 using gallic acid (y = 208604x + 173056; R2 = 0.999), compound 9 with myricetin (y = 23287x 581708; R2 = 0.999), compounds 10–11 with quercetin-3-O-glucoside (y = 34843x 160173; R2 = 0.999) and compounds 12–16 with peonidin-3-O-glucoside (y = 122417x 447974; R2 = 0.999). The results were expressed as mg/g of extract. 2.7. Bioactive in vitro assays 2.7.1. Antioxidant activity evaluation The lyophilized extracts were re-dissolved in ethanol:water (40:60, v/v) mixture to obtain a stock solution of 1 mg/mL, which were further diluted to obtain a range of concentrations for antioxidant activity evaluation by DPPH radical-scavenging, reducing power, inhibition of b-carotene bleaching and TBARS inhibition assays (Corrêa et al., 2015). The results were expressed as EC50 values (mg/mL), sample concentration providing 50% of antioxidant activity or 0.5 of absorbance in the reducing power assay. Trolox was used as positive control while the negative control was water. 2.7.2. Evaluation of cytotoxic properties The aqueous extracts were dissolved in water in order to obtain a final concentration of 8 mg mL 1. The final solution was further diluted to different concentrations (400–1.5 lg mL 1) to be subjected to in vitro cytotoxicity evaluation. Four human tumor cell lines were used: MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), HeLa (cervical carcinoma) and HepG2 (hepatocellular carcinoma), being all cell lines mycoplasma free. The cells were routinely maintained as adherent cell cultures in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) (MCF-7 and NCI-H460) and 2 mM glutamine or in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 2 mM glutamine, 100 U per mL penicillin and 100 mg per mL streptomycin (HeLa and HepG2 cells), at 37 °C, in a humidified air incubator containing 5% CO2. Each cell line was plated at an appropriate density [1.0  104 cells (10,000 cells) per well] in 96-well plates and allowed to attach for 24 h. The cells were then treated for 48 h with the different diluted sample solutions. Following this incubation period, the adherent cells were fixed by adding cold 10% TCA (100 lL) and incubated for 60 min at 4 °C. Plates were then washed with deionized water and dried; Sulforodamina B (SRB) solution (0.1% in 1% acetic acid, 100 lL) was then added to each plate-well and incubated for 30 min at room temperature. Unbound SRB was removed by washing with 1% acetic acid. Plates were air-dried, the bound SRB was solubilised with 10 mM Tris (200 lL, pH 7.4) and the absorbance was measured at 540 nm in the microplate reader mentioned above. The results were expressed in GI50 values (sample concentration that inhibited 50% of the net cell growth). For the negative control, cells were cultured with the same culture medium; however, the samples volumes were replaced by water (carrier solvent). Ellipticine was used as positive control. 2.7.3. Antibacterial activity evaluation The lyophilized samples were dissolved in water at 100 mg/mL and then submitted to further dilutions. The microorganisms used were clinical isolates from patients hospitalized in various departments of the Local Health Unit of Bragança and Hospital Center of Trás-os-Montes and Alto-Douro Vila Real, Northeast of Portugal. Six Gram-negative bacteria (Escherichia coli 1, E. coli 2, Klebsiella pneumoniae 1, K. pneumoniae 2, Morganella morganii and

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Pseudomonas aeruginosa isolated from urine and expectoration) and four Gram-positive bacteria (MRSA- methicillin-resistant Staphylococcus aureus, MSSA- methicillin-susceptible Staphylococcus aureus, Listeria monocytogenes and Enterococcus faecalis) were used to screen the antibacterial activity of the lyophilized extract. MIC determinations were performed by the microdilution method and the rapid p-iodonitrotetrazolium chloride (INT) colorimetric assay following the methodology suggested by Kuete, Ango, et al. (2011) & Kuete, Justin, et al. (2011) with some modifications. MIC was defined as the lowest extract concentration that prevented this change and exhibited inhibition of bacterial growth. Three negative controls were prepared (one with MHB/TSB, another one with the extract, and the third one with medium and antibiotic). One positive control was prepared with MHB and each inoculum. For the Gram-negative bacteria, antibiotics, such as amikacin, tobramycin, amoxicillin/clavulanic acid and gentamicin were used. For the Gram-positive bacteria, ampicillin and vancomycin were selected. The antibiotics concentrations applied are presented in the Supplementary Material. The antibiotic susceptibility profile of gram negative and gram positive bacteria has been already described by Dias et al. (2016). 2.8. Statistical analysis Three repetitions of the samples were used and triplicates for each concentration reading were carried out in all the assays. The results were expressed as mean values ± standard deviation (SD). The results were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s HSD Test with p = 0.05. For every parameter with only two available values a Student’s t-test was applied to determine the significant difference among the corresponding samples, with p = 0.05. When the p value was lower than 0.05, significant differences between samples were considered. Analyses were carried out using IBM SPSS Statistics for Windows, version 23.0. (IBM Corp., Armonk, New York, USA). 3. Results and discussion Although aware that the use of rat feces instead of human feces presents limitations, mainly due to the differences in microbiota (Becker, Kunath, Loh, & Blaut, 2011), this experimental model was chosen because both the anti-inflammatory and antioxidant effects of the hydroethanolic grape pomace extract have been assessed in a rat model in a recent work of our group (Gonçalves et al. 2017). Therefore, our attempt was to evaluate what would be the real molecules absorbed by the rats, reason why we made the option for the experimental model with animal feces. 3.1. Phenolic compounds analysis The samples were submitted to hydroethanolic extractions in order to obtain the largest possible number of compounds classes, reason why our samples were not acidified for anthocyanin stabilization purposes. Retention time, maximum absorption wavelengths in the visible region, mass spectral data and tentative identification of the Merlot grape pomace hydroethanol, digested and fermented extracts are show in Table 1. The phenolic profile of Merlot grape pomace hydroalcohlic extract was previously described by the authors, where the effect on the oxidative and inflammatory states of adjuvant-induced arthritic rats was investigated (Gonçalves et al., 2017). The most abundant phenolic compounds found in the three herein tested extracts were B-type (epi)catechin dimer (compound 2), (+)-catechin (compound 5) and ( )-epicatechin (compound 9) (Table 2). In the hydroethanolic extract, 20

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non-anthocyanin compounds were identified (with a total content of 66 mg/g of extract), a number that was significantly reduced to 11 compounds after the in vitro digestion. Apparently, compounds 7, 8, 11, 14, 16, 17, 19 and 20 were degraded during the digestion step, and remained absent from the extract after the simulated colonic fermentation. During the gastrointestinal digestion compound 2 had a reduction of almost 18-fold, while both compounds 5 and 9 suffered reductions around 4-fold. However, pronounced reductions in the contents of all phenolic molecules was apparent, being that the decreases were much more drastic after the in vitro digestion step, than after in vitro colonic fermentation. CorreaBetanzo et al. (2014), in their investigation over the stability and bioactivities of blueberry phenolic compounds during their passage through in vitro gastrointestinal digestion, also reported significant compound losses. The authors found that simulated intestinal digestion decreased both polyphenol and anthocyanin contents (by 49% and 15%, respectively) in comparison with the non-digested samples. During the simulated colonic fermentation (chemostat fermentation), some phenolic compound constituents suffered degradation (e.g., syringic, cinnamic, caffeic, and protocatechuic acids). According to the authors, the colonic fermentation also produced negative alterations in both antioxidant and antiproliferative potentials of blueberry phenolic compounds. Although the five anthocyanins identified in the hydroethanolic extract remained present in both digested and fermented extracts, after the simulated digestion process significant reductions occurred in their contents: compound 21 decreased 3-fold, compound 22 decreased in almost 7-fold, compound 23 decreased in more than 10-fold, and both compounds 24 and 25 presented reductions of almost 4-fold. On its turn, the in vitro colonic fermentation process apparently produced no significant losses of the anthocyanin compounds, once their contents remained practically unchanged after this last stage. Malvidin-3-O-glucoside (compound 23) was the most abundant anthocyanin in all three assessed extracts, followed by peonidin-3O-glucoside (compound 22). Ribeiro et al. (2015) identified thirteen anthocyanins in a hydroethanolic extract of Merlot grape pomace, within which all the five anthocyanins (compounds 21– 25) found in the present study were also included. The total anthocyanin content in the hydroethanolic extract (6.988 mg/g) was similar (8.280 mg/g) to the value reported by Rockenbach et al. (2011) for a Merlot grape pomace methanolic extract. Even though previous studies reported the presence of phenolic acids and valerolactones derivatives (Lingua, Fabani, Wunderlin, & Baroni 2016; Martins, Roberto, Blumberg, Chen, & Macedo, 2016; Gil-Sanches et al., 2017) in grape pomace samples, no peaks were found in the our extracts, whose UV spectra could be associated with phenolic acids, such as hydroxycinnamic acids or their tartaric or quinic esters (i.e., chlorogenic acids) or valerolactones derived from the digestion process. Further, no detection of those compounds could be made when the full mass chromatograms of the samples were screened for their molecular ions. Thus, no other phenolic compounds were identified in this extract, being characterized by the presence of flavonoids, mainly flavan-3-ols derivatives, galloyl derivatives, flavonols derivatives and five anthocyanins derivatives. 3.2. Evaluation of bioactive properties The in vitro antioxidant, antiproliferative, cytotoxicity and antibacterial properties of the Merlot grape pomace extracts, submitted or not to in vitro digestion and colonic fermentation were evaluated, and the results are presented in Table 3. For all four antioxidant activity evaluation assays, the hydroethanolic extract remained practically unchanged after the

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Table 1 Retention time (Rt), wavelengths of maximum absorption in the visible region (kmax), mass spectrometric data, and tentative identification of phenolic compounds of crude grape pomace, grape pomace submitted to in vitro digestion and grape pomace subjected to simulated colonic fermentation. Compounds

Rt (min)

kmax (nm)

[M H] (m/z)

MS2 (m/z)

Tentative identification

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

4.68 5.6 5.74 6.3 7.16 7.62 7.94 8.57 9.65 11.06 11.1 12.3 15.61 18.4 19.3 19.6 21.56 22.52 23.49 24.36

280 280 267 280 280 280 280 278 280 279 279 280 350 350 350 350 349 348 350 351

325 577 495 577 289 577 577 477 289 865 865 1153 479 477 463 493 433 447 477 655

169(100), 125(8) 451(23), 425(100), 407(22), 289(12), 287(10) 343(100), 191(8), 169(3) 451(23), 425(100), 407(22), 289(12), 287(10) 245(100), 203(50), 187(10), 161(9), 137(3) 451(23), 425(100), 407(22), 289(12), 287(10) 451(23), 425(100), 407(22), 289(12), 287(10) 325(100), 169(3), 125(2) 245(100), 203(35), 187(6), 161(8), 137(3) 739(78), 713(47), 695(100), 577(62), 575(42), 739(78), 713(47), 695(100), 577(62), 575(42), 865(25), 739(78), 713(47), 695(100), 577(62), 317(100) 301(100) 301(100) 331(100) 301(100) 301(100) 315(100) 509(15), 501(49), 475(63), 347(20), 329(100),

Galloylshikimic acid B-type (epi)catechin dimer Digalloylquinic acid B-type (epi)catechin dimer (+)-Catechin B-type (epi)catechin dimer B-type (epi)catechin dimer Digalloylshikimic acid ( )-Epicatechin B-type (epi)catechin trimer B-type (epi)catechin trimer B-type (epi)catechin tretramer Myricetin-O-hexoside Quercetin-3-O-glucuronide Quercetin-3-O-glucoside Laricitrin-O-hexoside Quercetin-O-pentoside Quercetin-O-rhamnoside Isorhamnetin-3-O-glucoside Methylisorhamnetin derivative

Compounds

Rt (min)

kmax (nm)

[M + H]+ (m/z)

MS2 (m/z)

Tentative identification

21 22 23 24 25

40.1 43.2 44.2 53.3 53.8

520 520 520 520 520

479 463 493 505 535

317(100) 301(100) 331(100) 301(100) 331(100)

Petunidin-3-O-glucoside Peonidin-3-O-glucoside Malvidin-3-O-glucoside Peonidin-3-O-acetylglucoside Malvidin-3-O-acetylglucoside

in vitro digestion. However, the simulated colonic digestion step promoted a significant decrease in the antioxidant capacities assessed by DPPH scavenging activity (6-fold reduction), b-carotene beaching inhibition (more than 2-fold reduction) and TBARS inhibition (6-fold reduction). Nonetheless, the antioxidant capacity data contained in Table 3, especially those regarding the hydroethanolic and digested extracts (EC50 values of 0.023 mg/mL and 0.029 mg/mL, respectively) assessed by TBARS formation inhibition, evidence a significant antioxidant potential of all Merlot grape pomace extracts tested herein, even after the in vitro colonic fermentation. Interestingly, only in the reducing power assay, it was observed a clear improvement of the antioxidant capacity (of almost 5-fold) after the colonic fermentation step. This result can be corroborated by the study of Del Pino-García et al. (2016), in which the authors verified that both in vitro gastrointestinal digestion and colonic fermentation promoted significant positive outcomes on the total antioxidant capacities of seasonings produced from red wine pomace. According to Pavan et al. (2014), the increment in the antioxidant activity of digested fruit extracts can be a result of phenolic compounds release after the in vitro digestion. Amico et al. (2008) found higher DPPH scavenging activity (EC50 = 0.01 mg/mL) for a Sicilian grape pomace hydroethanol extract than the values presented herein, while Otero-Pareja et al. (2015) reported an average EC50 of 0.008 mg/mL for grape pomace extracts from different varieties (including Merlot) obtained by pressurized liquid extraction using ethanol as solvent. On the other hand, Iora et al. (2015) reported lower antioxidant capacity for hydroethanol extracts of Merlot, Cabernet Sauvignon and Tanat grape pomaces, with DPPH_ assay values ranging from 5.05 to 6.54 mg/mL. Although the DPPH method is a unanimous choice in the evaluation of the antioxidant capacity of grape pomace (Fontana et al., 2013), the other methods used in the present study have not been much explored for this purpose. Hence, to the best of our knowledge, this is the first research work

425(12), 407(9), 289(6), 287(11) 425(12), 407(9), 289(6), 287(11) 575(42), 425(12), 407(9), 289(6), 287(11)

314(13)

on the antioxidant capacity of grape pomace using the herein set of antioxidant capacity methods. Results regarding the antiproliferative effects of the assayed extracts of Merlot grape pomace on the inhibition growth of four human tumor cell lines (MCF-7, NCI-H460, HeLa and HepG2) are shown in Table 3, expressed as concentrations that promoted 50% of the cell growth inhibition (GI50). In general, both hydroethanolic and digested extracts did not present antiproliferative activity against the tested tumor cell lines, except for the expressive cytotoxic activity (GI50=15 mg/mL) found for the grape pomace hydroethanol extract against the HeLa line (cervical carcinoma). However, the fermented grape pomace extract showed antiproliferative effects against all four tumor cell lines, with GI50 values ranging from 227 mg/mL (against HepG2) to 251 mg/mL (against HeLa). Jara-Palacios et al. (2015) found that a purified methanolic extract obtained from white grape pomace (Zalema variety), significantly inhibited adenocarcinoma cell proliferation (GI50 = 100 mg/mL), and also suggested that phenolic compounds contained in the extract (such as catechin and quercetin) were the mediating components of both anti proliferative action and direct initiation of cell death. Apparently, the in vitro colonic fermentation step promoted significant transformations that increased the bioactivity of the herewith tested Merlot grape pomace extract. Considering that both non-anthocyanin and anthocyanin compounds underwent significant degradation during the simulated digestion and fermentation steps, it can be inferred that the bioactive components responsible for the antiproliferative effects of the grape pomace are not the phenolic compounds shown in Table 1. In fact, other components of grape pomace extract, other than phenolic compounds, may be responsible for its observed bioactivities. All Merlot grape pomace extracts presented no toxicity in liver primary culture PLP2, being all the obtained GI50 values higher than the highest concentration tested (400 lg/mL) (Table 3). Ellipticine, the positive control, presented a GI50 of 2.29 lg/mL. The

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Table 2 Quantification of the identified phenolic compounds (mg/g extract) of crude grape pomace, grape pomace submitted to in vitro digestion and grape pomace subjected to simulated colonic fermentation. Compounds

Hydroethanol extract*

In vitro digestion

Colonic fermentation

Phenolic compounds non-anthocyanins Galloylshikimic acid B-type (epi)catechin dimer Digalloylquinic acid B-type (epi)catechin dimer (+)-Catechin B-type (epi)catechin dimer B-type (epi)catechin dimer Digalloylshikimic acid ( )-Epicatechin B-type (epi)catechin trimer B-type (epi)catechin trimer B-type (epi)catechin tretramer Myricetin-O-hexoside Quercetin-3-O-glucuronide Quercetin-3-O-glucoside Laricitrin-O-hexoside Quercetin-O-pentoside Quercetin-O-rhamnoside Isorhamnetin-3-O-glucoside Methylisorhamnetin derivative Total non-anthocyanin compounds

3.37 ± 0.04 10.2 ± 0.2a 2.30 ± 0.01a 6.7 ± 0.2a 7.27 ± 0.12a 3.2 ± 0.1a 5.33 ± 0.02 1.88 ± 0.03 7.3 ± 0.3a 4.9 ± 0.1a 3.6 ± 0.1 6.2 ± 0.3a 1.42 ± 0.01a 0.56 ± 0.02 0.52 ± 0.01a 0.36 ± 0.02 0.40 ± 0.01 0.38 ± 0.01a 0.51 ± 0.01 0.31 ± 0.01 66.6 ± 0.7a

nd 0.58 ± 0.02b 0.111 ± 0.002b 0.79 ± 0.01b 1.77 ± 0.01b 0.81 ± 0.01c nd nd 1.73 ± 0.02b 0.56 ± 0.02c nd 0.38 ± 0.01c 1.14 ± 0.01b nd 0.23 ± 0.01b nd nd 0.24 ± 0.01b nd nd 8.34 ± 0.05b

nd 0.48 ± 0.01c 0.113 ± 0.001b 0.85 ± 0.03b 1.44 ± 0.03c 0.90 ± 0.03b nd nd 1.53 ± 0.04c 0.89 ± 0.01b nd 0.67 ± 0.02b 1.11 ± 0.01c nd 0.22 ± 0.01c nd nd 0.23 ± 0.01c nd nd 8.42 ± 0.02b

Phenolic compounds anthocyanins 21 22 23 24 25 Total anthocyanin compounds

0.592 ± 0.001a 1.555 ± 0.002a 3.407 ± 0.001a 0.694 ± 0.001a 0.740 ± 0.001a 6.988 ± 0.003a

0.184 ± 0.001b 0.227 ± 0.002b 0.34 ± 0.01b 0.180 ± 0.001b 0.193 ± 0.002b 1.12 ± 0.01b

0.180 ± 0.001c 0.216 ± 0.003c 0.31 ± 0.01c 0.170 ± 0.0001c 0.181 ± 0.001c 1.06 ± 0.01c

nd – not detected. * Results previously published in Gonçalves et al. (2017). In each row different letters mean significant differences (p < 0.05).

Table 3 Antioxidant, cytotoxic, hepatotoxicity and antimicrobial activity of Merlot grape pomace hydroethanol extract, in vitro digestion and colonic fermentation (mean ± SD). Hydroethanol extract

In vitro digestion

Colonic fermentation

Antioxidant activity EC50 values (lg/mL) DPPH scavenging activity Reducing power b-carotene bleaching inhibition TBARS inhibition

58 ± 2b 101 ± 1b 215 ± 8c 23 ± 1c

60 ± 2b 158 ± 1a 257 ± 6b 178 ± 8a

365 ± 15a 34 ± 2c 599 ± 7a 29 ± 1b

Cytotoxic activity GI50 values (lg/mL)b MCF-7 (breast carcinoma) NCI-H460 (non-small cell lung carcinoma) HeLa (cervical carcinoma) HepG2 (hepatocellular carcinoma)

>400 >400 15 ± 1* >400

>400 >400 >400 >400

243 ± 7 242 ± 8 251 ± 9* 227 ± 6

Hepatotoxicity GI50 values (lg/mL)b PLP2

>400

>400

>400

Antimicrobial activity MIC values (mg/mL) Gram negative bacteria Escherichia coli ULSNE Escherichia coli CHTMAD Klebsiella pneumoniae ULSNE Klebsiella pneumoniae CHTMAD Morganella morganii Pseudomonas aeruginosa

20 >20 >20 >20 20 20

>20 >20 >20 >20 >20 >20

20 >20 >20 >20 20 >20

Gram positive bacteria Enterococcus faecalis Listeria monocytogenes MRSA MSSA

5 5 10 10

10 10 10 10

nd >20 10 20

a

EC50 values correspond to the sample concentration achieving 50% of antioxidant activity or 0.5 of absorbance in reducing power assay. GI50 values correspond to the sample concentration achieving 50% of growth inhibition in human tumor cell lines or in liver primary culture PLP2. a Trolox EC50 values: 62.98 lg/mL (DDPH), 45.71 lg/mL (reducing power), 10.25 lg/mL (b-carotene bleaching inhibition). b Ellipticine GI50 values: 1.21 mg/mL (MCF-7), 1.03 mg/mL (NCI-H460), 0.91 mg/mL (HeLa), 1.10 mg/mL (HepG2) and 2.29 mg/mL (PLP2). MIC values correspond to the minimal sample concentration that inhibited the bacterial growth. In each row different letters mean significant differences (p < 0.05). * Statistically different values, Student’s t-test p-value < 0.001.

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proved absence of cytotoxicity against liver cells, considered a great in vitro model for assessing human cytotoxicity (Corrêa et al., 2015), is a crucial requisite for the application of the tested extracts as nutraceuticals or food ingredients. Melo et al. (2015), which investigated the bioactivities of winery by-products, reported the low toxicity of hydroethanol extracts of Chenin Blanc, Petit Verdot and Syrah grape pomaces against mouse macrophage RAW 264.7 cells. The Merlot grape pomace extracts’ minimum inhibitory concentration (MIC) results for Gram-negative and Gram-positive bacteria are presented in Table 3. All three assessed extracts showed higher antibacterial efficiency against Gram-positive bacteria than Gram-negative bacteria, which corroborates the results reported by Tseng and Zhao (2012) in their work regarding the antimicrobial potential of Pinot Noir and Merlot grape pomaces. Our hydroethanolic extracts exhibited highest inhibitory activities against Enterococcus faecalis and Listeria monocytogenes, with significant reduction of their activities after the in vitro digestion (2fold reduction) and simulated colonic fermentation. Although the digestion and fermentation steps did not affect the inhibitory activity of the grape pomace hydroethanolic extract against MRSA-methicillin-resistant Staphylococcus aureus (MIC values remained 10 mg/ml), the in vitro fermentation did promote a 2fold reduction in its inhibitory activity against MSSA-methicillinsusceptible Staphylococcus aureus. Oliveira et al. (2013), when studying the bioactivities of Merlot grape pomace extracts obtained by CO2 supercritical extraction (SC-CO2), classified them as: strong inhibitors (MIC below 0.5 mg/mL), moderate inhibitors (MIC between 0.6 and 1.5 mg/mL) and weak inhibitors (MIC above 1.6 mg/mL). Based on their classification parameters, all grape pomace extracts assessed herein could be considered as weak inhibitors. Nevertheless, the bacteria strains used in this study are clinical isolated multiresistant strains (Dias et al., 2016), that present an antibiotic profile resistance much higher than ATCC bacterial strains. Oliveira et al. (2013) reported that the SC-CO2 Merlot grape pomace extracts were moderate inhibitors of Gram-positive bacteria (mainly S. aureus, with MICs ranging from 0.625 to 0.750 mg/mL) and weak inhibitors of Gram-negative bacteria (with MIC values above 1.6 mg/mL against E. coli and P. aeruginosa). Regarding the correlation between the bioactivity of the studied extracts and the presence of phenolic compounds, the results obtained showed slight correlation between peak 6 (B-type (epi)catechin dimer) and the antioxidant activity mearsured using the b-carotene bleaching inhibition and TBARS inhibition (R2=0.777 and 0.744, respectively) and peak 7 (B-type (epi)catechin dimer) with DPPH scavenging activity (R2=0.666); no correlation were observed for reducing power assay, cytotoxicity neither for antibacterial activity.

4. Conclusion Results of the present study showed that the in vitro digestion process led to drastic qualitative and quantitative reductions in the phenolic compounds profile of the Merlot grape pomace crude extract. Such alterations can be related to the decreases of some bioactivities of the extract, which seems to be the case of antioxidant and antibacterial properties, although not in a directly proportional manner. However, the in vitro digestion step apparently had no effect on the cytotoxic properties of the crude extract, except for the HeLa cell line. Interestingly, the simulated colonic fermentation seems to have a positive effect over the extract’s antiproliferative potential. Unquestionably, further in vivo studies such as dietary intervention, are necessary with the view to unravel and confirm these results. In order to reduce the losses of grape

pomace phenolic compounds and to preserve their bioactivities, the use of traditional and emerging microencapsulation technologies to ensure the delivery of these compounds could be easily performed. Overall, our findings contribute to the still scarce knowledge about the stability of grape pomace phenolic compounds and corresponding bioactivities during gastrointestinal digestion and colonic fermentation process, which could be useful in the development of nutraceutical supplements and functionalized food products. 5. Conflict of interests The authors declare no conflict of interests. Acknowledgments R.C.G. Correa thank Coordenação de Aperfeiçoamento do Pessoal do Ensino Superior (CAPES) and Fundação Araucária for the financial support provided for her post-graduate studies in Federal University of Technology - Paraná (contract 100/2014). Authors C. G. Kato and V.G. Correa thank CAPES for the financial support provided for their post-graduate studies in the State University of Maringá. R.M. Peralta (Project number 307944/2015-8) and C.W.I. Haminiuk (project number 303238/2013-5 and 304978/2016-7) are research grant recipients of Conselho Nacional de Desenvolvimento Científico e Tecnologia (CNPq). The authors are also thankful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Program PT2020 for financial support to CIMO (UID/AGR/00690/2013), L. Barros (SFRH/BPD/107855/2015) and M.I. Dias (SFRH/BD/84485/2012) grants. To POCI-01-0145-FEDER006984 (LA LSRE-LCM), funded by ERDF, through POCICOMPETE2020 and FCT. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jff.2017.07.030. References Amico, V., Chillemi, R., Mangiafico, S., Spatafora, C., & Tringali, C. (2008). Polyphenolenriched fractions from Sicilian grape pomace: HPLC–DAD analysis and antioxidant activity. Bioresource Technology, 99, 5960–5966. Barba, F. J., Zhu, Z., Koubaa, M., Sant’Ana, A. S., & Orlien, V. (2016). Green alternative methods for the extraction of antioxidant bioactive compounds from winery wastes and by-products: A review. Trends in Food Science & Technology, 49, 96–109. Becker, N., Kunath, J., Loh, G., & Blaut, M. (2011). Human intestional microbiota: Characterization of a simplified and stable gnotobiotic rat model. Gut Microbes, 2, 25–33. Bessada, S. M., Barreira, J. C. M., Barros, L., Ferreira, I. C. F. R., & Oliveira, M. B. P. P. (2016). Phenolic profile and antioxidant activity of Coleostephus myconis (L.) Rchb. f.: An underexploited and highly disseminated species. Industrial Crops and Products, 89, 45–51. Corrêa, R. C. G., de Souza, A. H. P., Calhelha, R. C., Barros, L., Glamoclija, J., Sokovic, M., et al. (2015). Bioactive formulations prepared from fruiting bodies and submerged culture mycelia of the Brazilian edible mushroom Pleurotus ostreatoroseus Singer. Food & Function, 6, 2155–2164. Corrêa, R. C., Peralta, R. M., Haminiuk, C. W., Maciel, G. M., Bracht, A., & Ferreira, I. C. (2016). The past decade findings related with nutritional composition, bioactive molecules and biotechnological applications of Passiflora spp. (passion fruit). Trends in Food Science & Technology, 58, 79–95. Correa-Betanzo, J., Allen-Vercoe, E., McDonald, J., Schroeter, K., Corredig, M., & Paliyath, G. (2014). Stability and biological activity of wild blueberry (Vaccinium angustifolium) polyphenols during simulated in vitro gastrointestinal digestion. Food Chemistry, 165, 522–531. Del Pino-García, R., González-SanJosé, M. L., Rivero-Pérez, M. D., García-Lomillo, J., & Muñiz, P. (2016). Total antioxidant capacity of new natural powdered seasonings after gastrointestinal and colonic digestion. Food Chemistry, 211, 707–714. Dias, M. I., Barros, L., Morales, P., Cámara, M., Alves, M. J., Oliveira, M. B. P., et al. (2016). Wild Fragaria vesca L. fruits: A rich source of bioactive phytochemicals. Food & Function, 7, 4523–4532.

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